1.1 Field of the Invention
The present invention provides adenosine deaminase (ADA) deficient mice. Also provided are methods of using the mice as an animal models for the analysis of physiological states that are sensative to disturbances in adenine nucleoside metabolism.
1.2 Description of Related Art
1.2.1 Adenosine Deaminase Deficiency
Genetic defects in purine metabolism in humans result in serious metabolic disorders, often with pronounced tissue-specific phenotypes (Blaese, 1995a). A striking example of this is adenosine deaminase (ADA) deficiency, which results in impaired lymphoid development and a severe combined immunodeficiency disease (SCID) (Hershfield and Mitchell, 1995).
ADA deficient SCID was the first of the inherited immunodeficiencies for which the underlying molecular defect was identified (Giblett et al., 1972); however, despite over 20 years of subsequent research, a satisfactory explanation for the lymphoid specificity of this metabolic disease has not emerged. This is largely due to the inaccessibility of human tissue for detailed phenotypic and metabolic analysis and the absence of an animal model which retains features of ADA deficiency in humans.
Additional interest in ADA deficiency stems from recent attempts to use novel therapeutic strategies, including enzyme therapy (Hershfield et al., 1993) and gene therapy (Bordingon et al., 1995; Blaese et al., 1995), to treat the condition in humans. Although the results of these therapeutic approaches are encouraging, unexpected outcomes have raised numerous important questions regarding the efficacy of specific treatment protocols (Hershfield et al., 1993; Blaese, 1995b). The pace with which new enzyme and gene therapy protocols can be tested would be greatly increased by the availability of an animal model for ADA deficiency.
The availability of a genetic animal model for ADA deficiency would make possible a wide range of biochemical and immunological experiments that are not permissible with humans.
Attempts to generate ADA deficient mice were initially reported by two groups (Wakamiya et al., 1995; Migchielsen et al., 1995), resulting in animals with independent sites of Ada gene disruption. However, these attempts did not lead to the production of viable ADA deficient mice. In each case a similar phenotype was observed. ADA deficient fetuses died perinatally due to severe liver damage (Wakamiya et al., 1995; Migchielsen et al., 1995). This phenotype was accompanied by profound disturbances in purine metabolism, including marked increases in the ADA substrates adenosine and 2'-deoxyadenosine.
2'-Deoxyadenosine is a cytotoxic metabolite that can kill cells through mechanisms that include disturbances in deoxynucleotide metabolism (Ullmann et al., 1978; Cohen et al., 1978) and the inhibition of cellular transmethylation reactions (Hershfield, 1979; Hershfield et al., 1979). ADA deficient fetuses exhibited evidence for both of these mechanisms of 2'-deoxyadenosine cytotoxicity, in that levels of the 2'-deoxyadenosine metabolite, dATP, were markedly elevated, and the enzyme S-adenosylhomocysteine (AdoHcy) hydrolase was inhibited (Wakamiya et al., 1995; Migchielsen et al., 1995). These metabolic disturbances are thought to contribute to the liver damage and subsequent death of ADA deficient fetuses.
Previous attempts by the inventors to produce mice that expressed ADA in the fetus but not in the neonatal mouse had failed (Blackburn et al., 1995). The strategy was to introduce an ADA transgene that would only be expressed in the placenta into heterozygous ADA knockout mice. The heterozygous transgenic mice could then be mated to yield homozygous ADA knockout mice that were able to develop because of the transgene provided ADA expression in the placenta. Viable mice were obtained by this strategy; however, the promoter used to express the transgene was not specific to the placenta and ADA expression was detected in the gastrointestinal tract of these animals, predominantly in the forestomach. Therefore, although ADA was not expressed in the lungs of these animals, ADA expression was present in the gut.
Disclosed herein is description of a mouse lacking post-partum ADA expression. This was acheived by creating a placenta specific promoter (Shi et al., 1997) and using this promoter in a transgene construct to express ADA. Indeed, the ADA deficient mice have immunodeficiencies similar to that of humans with ADA deficiency (Blackburn, 1998). Surprisingly and unexpectedly, the ADA deficient mice showed lung abnormalities. Upon extensive examination, the inventors were able to determine that the lung abnormalities in ADA deficient mice were reminiscent of those seen in asthma.
1.2.2 Asthma
Asthma is an inflammatory disease of the airways. In the U.S., 13% of children and 6% of adults suffer from asthma and 1% of health care cost are devoted to asthma treatment (Vogel 1997, Cochrane et al., 1996; Weiss et al., 1992). The disease is typified by the infiltration and activation of immune cells in the lung, followed by airway inflammation and obstruction (Vogel 1997). Many factors can trigger asthma, however, the mechanisms by which these triggers lead to airway inflammation and damage are not well understood. There is increasing evidence that asthma has its roots in early life (Barker 1992; Busse et al., 1995), but the mechanisms involved are not understood. Nor is it known how genetic and environmental influences interact to manifest asthma. Therefore, there is a need in the art for a model system with a predetermined genetic background that would allow testing of the influence of environmental factor on asthma development.
1.2.3 Animal Models and Asthma
Because of the limitations on the availability of human tissues, a number of animal models have been developed to better define the structural and functional consequences produced by environmental agents within the respiratory tract (Larsen and Colasurdo, 1997; Abraham and Baugh, 1995). While an ideal animal model should exhibit all the features of human asthma, there is general agreement that a single animal model does not exhibit all the functional and biological changes that would mimic the disease process seen in humans (Larsen and Colasurdo, 1997).
1.2.4 Adenosine Signaling in Asthma
Adenosine is a regulatory nucleoside that has the potential to be produced from all cells as a product of ATP catabolism (Arch and Newsholme, 1978). In mammalian tissues there exist two metabolic pathways through which adenosine can be generated: One is through the enzyme 5'-nucleotidase, which generates adenosine by enzymatic dephosphorylation of 5'-AMP (Zimmerman 1992). The other involves the hydrolysis of S-adenosylhomocysteine to adenosine and homocysteine (Schutz et al., 1981). Once generated, adenosine is freely transported in and out of cells through an ubiquitous nucleoside transporter (Arch and Newsholme, 1978) and serves as an extracellular signal. Inside the cell it is either deaminated to inosine by ADA (Blackburn and Kellems 1996) or is phosphorylated to AMP by adenosine kinase (Arch and Newsholme, 1978). Extracellular adenosine can bind to cell surface receptors to elicit a wide variety of cellular responses (Stiles 1992).
The regulatory actions of adenosine are mediated by several distinct membrane receptors that are classified as P1 purinergic receptors (Olah and Stiles 1995). Distinct subtypes of adenosine receptors have been identified on the basis of cDNA sequence and pharmacological profiles and are termed A1, A2a, A2b, and A3 adenosine receptors (Libert et al., 1989; Stehle et al., 1992; Pierce et al., 1992; Rivkees and Reppert, 1992; Fink et al., 1992; Zhou et a., 1992; Salvatore et al., 1993). Adenosine receptors are tightly coupled to effector enzymes by guanine nucleotide (G-protein) regulatory proteins (Stiles 1992; Olah and Stiles 1995), and each receptor subtype has a distinct affinity for adenosine and its structural congeners. Both A2 receptor subtypes couple to adenylate cyclase by stimulatory Gs-proteins and subsequently raise intracellular cAMP levels; in contrast, A1 and A3 receptors couple to inhibitory Gi-proteins and have the opposite effect (Olah and Stiles 1995). A1 and A3 receptors have also been shown to mediate the activation of potassium channels, inactivate calcium channels, stimulate or inhibit phosphatidylinositol turnover, inactivate phospholipase A2 or C, and inhibit chloride transport (Linden, 1991; Olah and Stiles 1995). Thus, adenosine signaling can have input into multiple intracellular signal transduction pathways. There is evidence that all four of these receptors are expressed in lung tissue and inflammatory cells and have all been implicated in participating in asthma (Dixon et al., 1996; Marquart 1997; Marquart et al., 1994).
Adenosine has been recognized as a potential signaling molecule in asthma (Holgate et al., 1992; Marquart 1997). Theophyline, an adenosine receptor antagonist, has been used in the treatment of asthma for decades (Barnes 1997). Adenosine is produced in response to tissue damage such as hypoxia (Arch and Newsholme, 1978) and has been shown to be released from hypoxic lung tissue (Mentzer et al., 1975), and to be elevated in bronchoalveolar lavage fluid (Driver et al., 1993) and blood (Mann et al., 1986) from asthma patients. Adenosine receptor expression increases in lung tissues from asthma patients (Richardson, 1997), and asthmatics are much more sensitive to exposure to adenosine than non-asthmatics (Cushley et al., 1983).
In vitro studies have shown that adenosine can enhance mediator release from human lung mast cells (Marquart et al., 1978), and adenosine itself can be released from stimulated mast cells (Marquart et al., 1984). Mast cell degranulation has been attributed to the A3 receptor (Reeves et al., 1997) and the A2b receptor has been implicated in IL-8 secretion from mast cells (Feoktistov and Biaggioni 1995). Adenosine also modulates pro-inflammatory events in eosinophils (Kohno et al., 1996; Walker 1996) and promotes macrophage differentiation (Najar et al. 1990). In addition, asthma induced in a rabbit model was prevented by preexposing the lungs to antisense oligonucleotides directed at blocking expression of the A1 receptor (Nyce and Metzer 1997).
Many of the known methods of treating asthma that are adenosine related focus on disrupting adenosine signalling through the receptors of the lung tissue. However, the present invention is the first to focus on lowering adenosine levels in an animal as a treatment for asthma. Surprisingly, the inventors were able to demonstrate that reduction of systemic adenosine levels alleviates asthma.
1.2.5 Adenosine and Deoxyadenosine Signaling in Severe Combined Immunodeficiency
ADA deficiency in humans is most often associated with a severe combined immunodeficiency (SCID) that is thought to arise from perturbations in signaling processes that result from the accumulation of the ADA substrates adenosine and deoxyadenosine (Hershfield and Mitchell, 1995). The exact mechanism through which accumulations in adenosine and/or deoxyadenosine lead to SCID are not know. Since the thymus and spleen, major immune organs, exhibit expression of adenosine receptors (Dixon et al., 1996), it is likely that adenosine signaling may be involved. Deoxyadenosine does not bind extracellular receptors, but has potent cytotoxic effects that are mediated through various intracellular pathways (Hershfield and Mitchell, 1995). Both adenosine and deoxyadenosine levels are elevated in the immune organs of the ADA deficient mice that are the topic of this invention 0Blackburn et al., 1998). In addition, these animals suffer from a combined immunodeficiency similar to that seen in ADA deficient humans. It is therefore likely that ADA deficient animals will serve as excellent models for determining how adenosine and deoxyadenosine signaling contribute to SCIDS, as well as serve as models for developing and testing therapies for the treatment of this disease, including enzyme therapy and gene therapy.
1.2.6 Adenosine Signaling in Other Physiological Systems
By generating animals that lack ADA, the enzyme responsible for controlling adenosine levels in tissues and cells, the inventors have created animals that exhibit systemic elevations in adenosine levels. It is therefore likely that these mice will prove useful in assessing the role of adenosine signaling in some if not all of these physiological systems. These systems include cardiovascular and vascular, neurologic, immunologic, renal, gastrointestinal, vascular, skeletal, and reproductive systems.